Evolvability describes an organism's capacity for producing descendants that are better adapted to a given environment. Mutations that elevate overall point mutation rates can make bacteria more evolvable under certain circumstances and consequently speed the progression of chronic infections and increase the incidence of antibiotic resistance. Little is currently known about how mutations affecting other cellular processes impact microbial evolvability or about how evolvability typically varies as microorganisms experience random mutations due to genetic drift or adapt by beneficial mutations to higher fitness. The proposed research systematically investigates how different kinds of mutations affect the evolvability of Escherichia coli in laboratory evolution experiments. The first aim is to test three strain series differing by predominantly deleterious, beneficial, or random mutations to establish baseline expectations. The second aim is to recover genotypes that eventually prevail over competitors of higher fitness because they are more evolvable by chronicling mutation dynamics in evolution experiments using deep sequencing, highthroughput genotyping, and deletion and microsatellite markers. The third aim is to construct chromosomal reporters with selectable markers for measuring gene amplification and deletion rates. These reporters will be used to isolate new kinds of genomic instability mutators and to examine whether a similar defect potentiated the evolution of a rare metabolic innovation in a 20-year evolution experiment. Throughout, evolvability will be measured on multiple time scales by comparing the initial divergence of marker trajectories in replicate evolution experiments to population genetic simulations and by performing co-culture competition assays between endpoint isolates and a reference strain. When a strain is found with unusually high or low evolvability, the causal mutation will be identified by genome re-sequencing, and its physiological consequences will be investigated to find out why it affects evolutionary potential.